UNIVERSITÀ DEGLI STUDI DI NAPOLI “FEDERICO II” PhD thesis “Marine organisms model species for the assessment of biological, environmental and economic impacts on marine aquaculture in Campania” Coordinatore Candidato Tutor Prof. Dott. ssa Dott. Giuseppe Cringoli Sara Fioretti Francesco Paolo Patti Prof. Aniello Anastasio “First, think. Second, believe. Third, dream. And finally, dare. If you can dream it, you can do it” Walt Disney Index Abbreviations list ......................................................................................13 Figures list .................................................................................................15 Tables list ...................................................................................................17 Abstract .....................................................................................................19 Introduction ..............................................................................................23 I Aquaculture in Campania: from the origin to the current times ..........25 II Aquaculture today: global and European trend ..................................27 III Aquaculture in Italy ...........................................................................30 IV Aquaculture in Campania ..................................................................31 V Mussel farms: breeding systems .........................................................33 VI Mussel farms threats and future perspectives for the Campanian industries ................................................................................................35 VII Introduction bibliography ................................................................38 Chapter 1 Mussels as model organisms ....................................................41 1.1 Model organisms ..............................................................................43 1.2 Bivalves as model organism .............................................................45 1.3 The genus Mytilus as model organism .............................................47 1.3.1 Mytilus galloprovincialis and Mytilus unguiculatus Valenciennes ......................................................................................49 1.4 Mussels provide ecosystem services ................................................55 1.4.1 Threats to ecosystem services ...................................................57 1.4.2 Food frauds ................................................................................59 1.4.3 Ocean Acidification ...................................................................61 1.5 Chapter 1 bibliography .....................................................................65 Chapter 2 Sea food frauds: a threat for mussels economy ........................77 2.1 Bivalves as food product ..................................................................79 2.1.1 Seafood labels ............................................................................81 2.2 Seafood frauds ..................................................................................84 2.2.1 Health, economic and environmental threats ............................85 7 Index 2.2.2 DNA barcoding and other molecular techniques for seafood frauds identification ............................................................................87 2.3 Mussels threatened by seafood frauds ..............................................91 2.3.1 The situation in Italy and in Campania .....................................92 2.4 Enhance Campanian mussels and their local market .......................95 2.4.1 Sampling area ............................................................................96 2.4.2 DNA extraction .........................................................................99 2.4.3 Molecular markers ...................................................................100 2.4.3.1The molecular marker COI ................................................102 2.4.3.2 The molecular marker 16S ................................................102 2.4.3.3 The molecular marker PAPM ............................................103 2.4.3.4 The molecular marker 28S ................................................104 2.4.4 Purification of PCR products, sequencing and molecular software analisys ..............................................................................104 2.4.4.1 The assessment of Mytilus galloprovincialis species ........105 2.4.4.2 The attempt in local mussel origin identification ..............105 2.5 Our findings, our fish market, our mussels ....................................113 2.6 DNA extraction protocol for molluscs ...........................................118 2.7 Chapter 2 bibliography ...................................................................119 Chapter 3 Anthropogenic stressors: mussels responses to Ocean Acidification .............................................................................................129 3.1 Ocean Acidification: molluscs responses .......................................131 3.1.1 Mussels responses to OA ........................................................134 3.2 OA effects on Mytilus unguiculatus Valenciennes ........................137 3.2.1 Experimental setup ..................................................................137 3.2.2 Genetic characterization ..........................................................140 3.2.3 Physiological parameters: oxygen consumption (MO2), + ammonia (NH4 ) excretion and O:N ratio ........................................140 3.2.4 Expression of NKA (Na+-K+-ATPase alpha subunit) and NHE8 (Sodium 8 hydrogen exchanger) genes ............................................144 8 Index 3.2.5 UPLC analysis .........................................................................149 3.2.6 Shell contents of Ca2+ and Mg+ ...............................................153 3.2.7 Shell structure ..........................................................................154 3.2.8 X-ray testing ............................................................................155 3.2.9 Statistics……………… ……………………………………..155 3.3 OA effects on Mytilus unguiculatus Valenciennes: a global overview ..............................................................................................................159 3.4 RNA extraction protocol ................................................................166 3.5 Amino acid analysis .......................................................................168 3.6 Chapter 3 bibliography ...................................................................170 Chapter 4 A biotechnological application: can Mytilus galloprovincialis produce a pearl? ........................................................................................179 4.1 Pearls, fascinating creations made by molluscs .............................181 4.2 Pearl formation ........................ Errore. Il segnalibro non è definito. 4.3 Pearl industry ........................... Errore. Il segnalibro non è definito. 4.3.1 Grafting ............................ Errore. Il segnalibro non è definito. 4.3.2 Different kind of pearls ..... Errore. Il segnalibro non è definito. 4.4 Can Mytilus galloprovincialis made a pearl? ... Errore. Il segnalibro non è definito. 4.4.1 Different nucleus materials and the “classical grafting” .. Errore. Il segnalibro non è definito. 4.4.2 New grafting technique .... Errore. Il segnalibro non è definito. 4.4.3 Campanian pearls: new marketable products? ............. Errore. Il segnalibro non è definito. 4.5 Chapter 4 bibliography ...................................................................201 Chapter 5 Mussels market in Campania: anchor in the history and exiting future perspective .....................................................................................205 5.1 Mussels in the Bourbons kitchen ....................................................207 5.2 Mussels in our kitchen ....................................................................210 5.2.1 Campanian Mytilus galloprovincialis mussel as an excellent product ..............................................................................................212 9 Index 2.2.2 Will mussels still exist in our future kitchen? .........................214 5.3 The interesting mussels perspectives outside the kitchen ..............217 5.3.1 Mussels as pharmaceuticals, functional foods, food ingredients and much else ...................................................................................217 5.3.2 Campanian pearls ....................................................................220 5.4 The end of this project but not of the story…. Mytilus galloprovincialis: a positive Campanian aquaculture model ...............223 5.5 Chapter 5 bibliography ...................................................................225 10 Acknowledgemetns Thanks to my tutor, the doctor Francesco Paolo Patti. Thanks for the great opportunity that offered me, for being my “daddy in science” since the master thesis. Thank you for teaching me so many things and supporting me even when my creativity gained the upper hand…! Thanks to the professor Aniello Anastasio, my university tutor. Thanks to all the staff of the Stazione Zoologica Villa Dohrn- Benthic Ecology Center, in Ischia. You have all been a precious and fundamental presence, for sampling, for your technical help, for your friendship. Thanks to the professor Yung-Che Tseng for hosting me in his beautiful Lab of Marine Organismic Physiology in